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WO2007069681A1 - Kit de dosage de l’ingredient actif d’un agent anti-termites au moyen d’un procede d’immunodosage - Google Patents

Kit de dosage de l’ingredient actif d’un agent anti-termites au moyen d’un procede d’immunodosage Download PDF

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Publication number
WO2007069681A1
WO2007069681A1 PCT/JP2006/324930 JP2006324930W WO2007069681A1 WO 2007069681 A1 WO2007069681 A1 WO 2007069681A1 JP 2006324930 W JP2006324930 W JP 2006324930W WO 2007069681 A1 WO2007069681 A1 WO 2007069681A1
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WO
WIPO (PCT)
Prior art keywords
active ingredient
reaction
control agent
termite control
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2006/324930
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English (en)
Japanese (ja)
Inventor
Shiro Miyake
Mikiko Uchigashima
Atsushi Kadowaki
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Horiba Ltd
Bayer CropScience KK
Original Assignee
Horiba Ltd
Bayer CropScience KK
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2006309332A external-priority patent/JP2007187650A/ja
Priority claimed from JP2006332073A external-priority patent/JP2008145240A/ja
Application filed by Horiba Ltd, Bayer CropScience KK filed Critical Horiba Ltd
Priority to AU2006325990A priority Critical patent/AU2006325990B2/en
Priority to EP06834684A priority patent/EP1965209A4/fr
Priority to US12/097,627 priority patent/US8323904B2/en
Publication of WO2007069681A1 publication Critical patent/WO2007069681A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/10—Insecticides

Definitions

  • the present invention relates to a kit for measuring termite control agents using an immunoassay. It is useful for a simple measurement kit of termite control concentration such as imidacloprid, which remains in the soil, such as fiprole! /, Particularly for measurement at the application site of termite control agent.
  • a simple measurement kit of termite control concentration such as imidacloprid, which remains in the soil, such as fiprole! /, Particularly for measurement at the application site of termite control agent.
  • Termite repellents are applied or dispersed to the foundation or floor to construct a target building, etc., and then must have a long-lasting effect in soil etc. Monitoring is required.
  • the active ingredient form of the termiticide is contained in an emulsion etc., it may be contained in a capsule preparation such as a microcapsule.
  • the residual concentration of the active ingredient in a termite repellent has been analyzed by gas chromatography (GC) after extracting and purifying the component of the target soil equivalent force repellent. That is, the sample is extracted with an organic solvent and purified sequentially by a plurality of columns such as a porous silica column, a florisil column, a silica gel column, a C column, and a florisil column.
  • GC gas chromatography
  • an immunoassay measures an antigen by utilizing specific reactivity of the antibody to the antigen, and such a complex GC like the above-mentioned GC which is superior in measurement accuracy Because it does not require purification processes or expensive equipment, it is a fast, simple and economical measurement method.
  • immunoassays have played a large role as one of patient condition analysis methods in the field of clinical diagnosis, but in recent years their application to measurement of environmental impact chemicals has been advanced.
  • termite control agents several types have developed immunoassays for active ingredients of termite control agents using monoclonal antibodies prepared using as a immunogen a complex of a derivative of the active ingredient and a protein bound thereto. (E.g., JP-A-2000-191698).
  • Substances used as an active ingredient of termite control agents include organophosphorus insecticide chlor There may be mentioned Pleurothion, pyridafenthion, penoremethrin of pyrethroid series, trarometricin, creosote oil of a mixture of taresol and naphthalene, fipronil of pyruvirazole series and imidacloprid of neonicotinoid series.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2000-191698
  • immunoassay is a measurement method relatively suitable for on-site measurement because it does not require complicated purification steps.
  • immunoassay methods using microplates that are commonly used are also suitably used in analysis facilities, and are suitable for use in residential areas where termite control agents are applied. To provide you with a powerful force
  • the active ingredient is present as a capsule preparation such as a microcapsule
  • measurement can not be made unless the capsule part is dissolved, and a more convenient measurement method at the application site has been required.
  • An object of the present invention is to provide a method for measuring termite eliminants having excellent operability using an immunoassay and a kit therefor in the measurement at the application site of the termite repellent.
  • the present invention is a kit for measuring the active ingredient of a termite control agent by immunoassay.
  • 1) Measurement subject force Extracting unit for extracting the active ingredient of termite repellent using solvent, 2) Reaction container for enclosing the identification antigen, fixed member for immobilizing the anti-active ingredient antibody, and the above-mentioned reaction container It is an object of the present invention to provide a kit for measuring an active ingredient of a termite eliminator using an immunoassay, which has a reaction unit, and a sealing member which can be fitted to the
  • the identification antigen is a conjugate of a hapten of the active ingredient of the termite control agent and a substance having a identification function.
  • the antibody may be a monoclonal antibody or a fragment thereof.
  • the kit for measuring an active ingredient of termite control agent using the immunoassay method of the present invention as the above reaction container, at least one reaction container in which the identification antigen is enclosed, the identification antigen and the known amount Combinations with at least one reaction vessel containing the active ingredients of termite control can be used.
  • the above-mentioned reaction in a state where the above-mentioned identification antigen and the identification antigen and a mixture of an identification ingredient and a known amount of termite control agent are dried beforehand While being enclosed in a container, it can be dissolved with the sample solution in a reaction container containing only the identification antigen, and can be dissolved with a solution in a reaction container in which the mixture is sealed.
  • the reaction unit visually or optically detects a change depending on the concentration of the active ingredient of the termite control agent in the sample. It can also serve as a detection function for detection.
  • a kit for measuring an active ingredient of a termite control agent using the immunoassay method of the present invention is a detection unit for visually or optically detecting a change depending on the concentration of the active ingredient of the termite control agent in the above sample. Can also be included.
  • the kit for measuring an active ingredient of a termite control agent using the immunoassay method of the present invention includes a dilution unit for diluting the sample solution extracted by the above extraction unit to a predetermined ratio.
  • a solvent can be included in the above extraction unit.
  • This solvent is ethanol, methanol And one or more selected from the group consisting of dimethyl sulfoxide and dimethyl sulfoxide.
  • the above-mentioned measurement target may be a capsule formulation inclusion.
  • the capsule formulation may be in the form of microcapsules.
  • the method of measuring the active ingredient of the termite control agent by the immunoassay method of the present invention comprises the steps of: 1) extracting the active ingredient of the termite control agent from the collected measurement object using a solvent; Contacting the sample solution containing an active ingredient of the termite control agent obtained in the step with a fixing member on which the antibody against the active ingredient is immobilized in a reaction container in which the identification antigen is encapsulated; Detecting an amount of change depending on the concentration of the active ingredient of the termiticide in the sample, which is caused by an antigen-antibody reaction.
  • the identification antigen is a conjugate of a hapten of the active ingredient of the termite control agent and a substance having a identification function. May be a monoclonal antibody or a fragment thereof.
  • the step of detecting may be a step of comparing the amount of change in the sample solution obtained by the extraction with the amount of change in the sample solution containing the active ingredient of a certain amount of termite control agent.
  • the active ingredient of the termiticide can be fipronil or imidacloprid.
  • fiprole (a scientific name: 5 amino- 1-(2, 6 dichloro- 1 4- (trifluoromethyl) phenyl)-4 ((tri- fluoromethyl) sulfeel) 1 H pyrazole-3- carbo -Toll) is the following formula (1):
  • SOCF 3 In addition to being used as a termiticide, it has the structure represented by and as insecticides, insecticidal agents such as dung beetles, aphids, whiteflies, stink bugs, mealybugs, pterocarp, etc. It exhibits excellent efficacy against a wide range of agricultural pests such as lepidopteran pests such as leopard insects, moths, moth moths, moth moths, moth moths, mikanno, mogriga, etc.
  • insecticidal agents such as dung beetles, aphids, whiteflies, stink bugs, mealybugs, pterocarp, etc. It exhibits excellent efficacy against a wide range of agricultural pests such as lepidopteran pests such as leopard insects, moths, moth moths, moth moths, moth moths, mikanno, mogriga, etc.
  • imidacloprid (chemical name: 1-(6-(3)-6-pyridylmethyl) N--toroimidazazolidine-2-ylideneamine)) has the following formula (2):
  • the present compounds are insecticidal agents such as dung beetles, aphids, psyllids, bugs, worms, worms and the like. It is effective against lepidopteran pests such as kinmon hosoga, ganmon hamoguriga, peachhamogriga, mandarinammoriga, etc., and also broad-leaved agricultural pests such as coleopteran pests and orthopteran pests, and has penetration transfer and residual efficacy.
  • lepidopteran pests such as kinmon hosoga, ganmon hamoguriga, peachhamogriga, mandarinammoriga, etc.
  • broad-leaved agricultural pests such as coleopteran pests and orthopteran pests, and has penetration transfer and residual efficacy.
  • FIG. 1 An explanatory view showing a specific example of a reaction container according to the present invention.
  • FIG. 2 Standard curve obtained by measuring fipronil with the measurement kit according to the present invention.
  • FIG. 3 Standard curve obtained by measuring imidacloprid with the measurement kit according to the present invention.
  • An immunoassay using an antigen-antibody reaction generally has high selectivity to a specific substance, is capable of highly sensitive measurement, and is an operation easy to operate.
  • the first step is to introduce the prepared sample into the reaction container in which the identification antigen is sealed, thereby eliminating termite in the sample. Uniform mixing of the active ingredient of the agent with the identification antigen can be achieved.
  • termite control is to insert or introduce into the reaction container a carrier on which an antibody against the active ingredient of the agent is immobilized, as a second step, thereby providing a competitive antigen-antibody reaction between the active ingredient of the termite control agent and the identification antigen. Can be done quickly and reliably.
  • the reaction product obtained by such reaction is detected as a coloring reaction derived from the discrimination antigen bound to the antibody. By optically detecting these changes, the concentration of the active ingredient of the termiticide can be determined.
  • These competitive reactions and color reactions can be performed in a closed state so that they can be easily operated outdoors.
  • the reaction container in which the identification antigen is sealed in advance has a feature of performing all operations such as stirring, reaction, and measurement.
  • detecting visually or optically refers to the color change or fluorescence generated by the reaction itself or the color change generated by the addition of a color former or a fluorescent agent. It means taking out as optical change such as fluorescence or turbidity, and detecting it visually or by absorption spectrophotometer.
  • the identification antigen has a molecular structure close to the active ingredient of the termite control agent and an antibody. It is preferable to use as the identification antigen a substance having an epitope of In the present invention, this condition can be satisfied and measurement with high selectivity can be made by using the identification antigen as a conjugate of a derivative of the active ingredient of the termiticide and a substance having a identification function.
  • the "substance having a discrimination function” refers to a substance or carrier substance which makes it possible to discriminate the result of the antigen-antibody reaction directly or indirectly, and more specifically, a biological substance such as an enzyme or a fluorescent protein And low molecular weight fluorescent substances, gold colloids, and carriers such as colored latex beads. Moreover, in the antigen-antibody reaction according to the present invention, not only such an antibody but also a part of an antibody having an antigen binding property such as a Fab fragment or an F (ab,) 2 fragment is included.
  • the active ingredient of the termite eliminator is creosote oil of a mixture of chlorpolis, huetrothion, pyridafenthione, permethrin, tralomethrin, taresol, naphthalene, etc., oil, fiprole, imidacloprid, thiamethoxam, crothiazine, dinotefuran etc. It may be any compound of In order to detect these compounds, the respective compounds are used as they are or their derivatives are bound to a labeling substance and used in the immunoassay of the present invention.
  • the active ingredient of the termite inhibitor in the sample solution is detected. It is preferable to clarify the criteria for the concentration of Therefore, by preparing multiple reaction vessels containing the active ingredients of termite control agent of known concentration including zero, and comparing the change in color amount with the reaction vessel into which the sample liquid was added, it is possible to It is possible to make measurements without the influence of That is, by comparing the measurement conditions with a common reference, it is possible to provide a kit or a method for measuring the active ingredient of the white-wing control agent using a simple and highly accurate immunoassay.
  • Some drugs generally used as identification antigens may lack long-term stability when left to stand. When such alteration occurs, it affects not only the reaction rate but also the reaction itself as the activity decreases, which may greatly affect the measurement accuracy.
  • the present invention has been found that by lyophilizing the identification antigen, it is possible to prevent deterioration of the drug and to ensure an optimal reaction state immediately before use.
  • a liquid hereinafter referred to as “sample liquid” or a solution
  • an antigen-antibody reaction is initiated to measure the active ingredient of the termite control agent using a rapid immunoassay. It becomes possible.
  • the reaction can be initiated and completed in the reaction vessel, it is possible to construct a measurement kit excellent in on-site operability.
  • the term "lysing solution” as used herein does not take part in the reaction, for example, dilution solution, and dissolves a mixture of the identification antigen or identification antigen that has been lyophilized in advance and the known ingredient of the termite active ingredient and a known amount.
  • visual or optical change detection such as color change may be performed in the reaction unit as a result after the antigen-antibody reaction, or may be performed by providing another detection unit. It can also be done.
  • this dilution kit that includes a dilution unit for diluting the sample solution containing the effective components of the termite control prepared in the extraction unit to a predetermined ratio.
  • the diluted sample solution may be diluted to an appropriate concentration for the reaction.
  • the solvent contained in the extraction unit may be one or a combination of two or more selected from the group consisting of ethanol, methanol and dimethyl sulfoxide.
  • the measurement target contains a capsule preparation, which may be in the form of microcapsules.
  • the termite repellent such as an effective component of termite repellent is enclosed in a microcapsule and used. That is, while the effects of termite control agents on the human body are minimized by the capsule, at the same time, for the termites, the microcapsules in the mouth are squeezed and crushed by dulling etc. The active ingredient in the microcapsules exerts its effect. For this reason, recent years have widely used microcapsule technology including microcapsules. Therefore, it is necessary to take measures to accurately measure the amount of termite control active ingredient in the capsule preparation, and it is necessary to select a solvent that dissolves the capsule part in the sample and extracts the active ingredient of termite control agent. Is important.
  • the kit for measuring the active ingredient of a termite control agent using the immunoassay method according to the present invention (1) Strength of the collected measurement object: Extraction of active ingredient containing termite control agent extracted using a solvent for preparation of a sample liquid Unit, (2) reaction container for encapsulating the identification antigen, fixing member for immobilizing the active ingredient antibody of antitermite pesticide, and sealing member which can be fitted to the reaction container, and the sample liquid A reaction unit characterized by conducting an antigen-antibody reaction by contacting the
  • the measuring method of the above-mentioned measuring kit may be any of known immunoassays, for example, enzyme immunoassay The epidemic assay method, the gold colloid method etc. (Meth. EnzymoL, 92, 147-523 (1983), Antibodies Vol. 11 IR L Press Oxford (1989 ⁇ ⁇ ⁇ ) can be applied.
  • each unit serving as the basic composition of the assay kit will be described, taking as an example the case where an antibody solid phase ELISA (Enzyme-Linked Immunosorbent Assay) is applied as an example. Is not limited to this.
  • each material to be described is not limited to the measurement kit to which the ELISA is applied, but is commonly used in the measurement kit when other label is used, or the measurement method when the ELISA or other label is used. It is possible.
  • examples of the object to be measured include soil and environmental water, and as the substance to be measured, the effective components of termite repellents are targeted.
  • the soil is added to a container made of polypropylene, a resin such as polyethylene containing polyethylene or the like, or a glass container, and the active ingredient of the termiticide is extracted by stirring.
  • solvents for extraction include methanol, acetone, acetonitrile, ethyl acetate, ethanol, dimethyl sulfoxide, and dimethylformamide.
  • solvents may be pre-packaged or separately included in the kit. Or the kit contains only the container, and the solvent can be procured separately.
  • the preferred concentration of solvent is about 30 to 100%. That is, these solvents may be in the form of an aqueous solution.
  • the solvent is preferably, but not limited to, one capable of dissolving the capsule part even if the active ingredient of the pesticide is present in the form of a capsule preparation such as a microcapsule. That is, a solvent that can be used regardless of the formulation form of the active ingredient of the termiticide is preferred.
  • Methanol is excellent in that it can obtain high extraction efficiency with high solubility in the termite repellent active ingredient. In addition, they are also excellent in that they are less denatured to proteins with low hydrophobicity. Furthermore, it is more preferable because methanol can obtain high extraction efficiency by 40 to 50% and the risk is small.
  • any of the above solvents can be used to completely dissolve the capsule component.
  • Ethanol or dimethylsulfoxide is preferred.
  • ethanol is a relatively less toxic commercially available anhydrous ethanol. It is preferable because it can be used for simple measurement.
  • an active ingredient or its derivative of the antiviral substance, which is bound to the enzyme is used as a derivative. It is preferable to use one having a binding group.
  • the enzymes that can be used are not particularly limited as long as they are known, including peroxidases, alkaline phosphatase, j8 galactosidase and the like.
  • the combination of the derivative and the enzyme may be performed by any method without particular limitation as long as the conditions do not inactivate the enzyme.
  • the structures of the derivatives of fiprole and the derivatives of imidacloprid will be specifically described below in relation to the effective components of the termiticide, but the present invention is not limited thereto.
  • Alpha is - S (0) m-, oxygen atom, - CH- and - NH- selected from the group forces also
  • L is one selected from the group consisting of a carboxyl group, an amino group, an aldehyde group and a hydroxyl group, m is an integer of 0 to 2 and n is an integer of 1 to 10.
  • a complex is formed by covalent bonding of L to a target polymer.
  • the oxidizing agent is not particularly limited in this reaction, and for example, peroxy acids such as hydrogen peroxide, peracetic acid, perbenzoic acid, metachloroperbenzoic acid, ozone, potassium permanganate, chromic acid, etc. are used. be able to. Also, tungsten or vanadium can be used as a catalyst.
  • the solvent include alcohols such as methanol and ethanol, aromatic hydrocarbons such as benzene and toluene, ethers such as jetyl ether, dipropyl ether and tetrahydrofuran, acetone, methyl ethyl ketone and the like.
  • Ketones acetonitrile, tolyl such as propio-tolyl, acid amide such as dimethylformamide, dimethyl acetoamide, sulfoxide such as dimethyl sulfoxide, acid such as acetic acid, water, mixed solvents thereof Can be mentioned.
  • the reaction temperature is usually from room temperature to the boiling point of the solvent, for about 30 minutes to about 10 hours.
  • Step 1 As the base and solvent used in this reaction, the same ones as in Step 1 can be used.
  • the reaction is usually carried out at a temperature of 0 ° C., at the boiling point of the solvent, and for about 30 minutes to 10 hours.
  • Examples of derivatives of imidacloprid include compounds having a structure represented by the following formula (5).
  • A represents one selected from the group consisting of S, 0, CH and NH, and n is 1
  • a complex is formed by covalently bonding a carboxyl group to a target polymer to a compound represented by the above-mentioned formula (5).
  • the production of the compound represented by the above-mentioned formula (5) used as a hapten compound can be carried out by a known synthesis method and is not particularly limited. For example, the following method Can be used.
  • L 2 is a halogen atom selected from Cl, Br, and a group which is also an I force
  • a halogen atom selected from Cl, Br, and a group which is also an I force are selected from compounds having the structure shown below in an organic solvent in the presence of a base
  • the protective group for carboxyl group represented by P is a known one, and specific examples thereof include methyl, ethyl, tert-butyl, benzyl, p-methoxybenzyl and 3, 4-di Examples thereof include a methoxybenzyl group, a trichloroethyl group, a trimethylsilyl group, a tert-butyldimethylsilyl group, a tert-butyl diphenylsilyl group, a triethylsilyl group, a triisopropylsilyl group, and a trimethylsilylethyl group.
  • the reaction is carried out at a temperature of 0 ° C. to the boiling point of the solvent, preferably 10 ° C. to 100 ° C., for 5 minutes to 10 hours, preferably 30 minutes to 2 hours.
  • the solvent examples include, for example, methanol, ethanol, benzene, toluene, xylene, dichloromethane, chlorofone reme, carbon tetrachloride, jetinole tenore, tetrahydrofuran, dioxan, acetone, methylethyl ketone, acetonitrile, ethyl acetate Dimethylformamide, dimethylsulfoxide, water and the like can be used.
  • sodium carbonate And potassium carbonate sodium hydroxide, potassium hydroxide, sodium methylate, sodium ethylate and the like.
  • the reduction reaction can be performed using a known method. For example, using a reducing agent such as sodium borohydride or lithium lithium aluminum in a solvent such as methanol, ethanol, benzene, toluene, xylene, jetyl ether, tetrahydrofuran, dioxane, acetylene, ethyl acetate, acetic acid and water. Do.
  • the reaction is carried out with stirring at a temperature of -80.degree. C. and also at the boiling point of the solvent, preferably 0.degree. C. to 50.degree. C., for 5 minutes to 10 hours, preferably 30 minutes to 5 hours.
  • L 3 is a halogen atom from which a group force which is also a Cl, Br, and I force is also selected; and A, P and n are as defined above]
  • the reaction is carried out at a temperature of 0 ° C. to the boiling point of the solvent, preferably at room temperature to 100 ° C., for 5 minutes to 10 hours, preferably for 30 minutes to 3 hours.
  • the compound of the formula (5) can be obtained by removing the carboxyl-protecting group represented by P from the compound of the formula (X7).
  • the removal of the protecting group of the carboxyl group can be carried out by known methods such as alkaline hydrolysis, acid hydrolysis and the like.
  • the compound of the formula (X7) is preferably dissolved in an organic solvent such as acetic acid, formic acid, benzene, dichloromethane, 1,2-dichloroethane and the like, and then hydrochloric acid, sulfuric acid, C. Boron trifluoride jetyl ether complex, trifluoroacetic acid, trifluoromethanesulfonic acid, p-toluenesulfonic acid, etc. are added at 0.degree. C. power boiling point of solvent, preferably at 0.degree. C. to 50.degree. C.
  • the compound of the formula (2) can be obtained by stirring and reacting for 5 minutes to 10 hours, preferably 1 hour to 5 hours.
  • the compound of the formula (X7) is preferably dissolved in an organic solvent such as methanol, ethanol, tetrahydrofuran or ethylene glycol, and then sodium hydrogen carbonate, sodium carbonate, potassium carbonate, Add lithium hydroxide, sodium hydroxide aqueous solution or potassium hydroxide aqueous solution, etc., and heat at the boiling point of the solvent at 0 ° C, preferably 0 ° C for 5 minutes to 10 hours, preferably 1 hour
  • the compound of the formula (1) can be obtained by reacting for 2 hours with stirring.
  • the removal can also be carried out by hydrogenolysis with hydrogen.
  • deprotection can also be carried out with a reagent that generates fluorine ions such as tetra-n-butyl ammonium fluoride, pyridinium fluoride and the like.
  • the complex obtained by the above-mentioned production method can be made into a purified product of higher purity by performing silica gel chromatography or recrystallization operation as necessary.
  • the immune reaction container is a container made of a resin such as polypropylene or polyethylene or a glass. It is preferred to use. In addition, it is preferable that the present reaction container has a structure capable of inserting a sealing member on which an antibody is immobilized, but is not limited thereto.
  • the reaction container described above preferably uses a combination of at least one reaction container in which the identification antigen is previously encapsulated and at least one reaction container in which the identification antigen and the known amount of the active ingredient are previously encapsulated.
  • the concentration of the active ingredient in the sample is detected as a change in color amount after the color reaction, the criteria for the change in color amount such as the background state or the reaction result by the active ingredient of known concentration are clarified. It is preferred to determine the concentration by comparison with them.
  • a sample solution containing an active ingredient is put into one reaction container in which a discrimination antigen is previously encapsulated, and one or more of the discrimination antigen and a known amount (in the case of two or more) are introduced.
  • the concentration of the active ingredient in the sample solution can be determined from the relationship with the reference known amount of the active ingredient by comparison with the reaction container in which the mixture consisting of different known quantities of the active ingredient is enclosed. it can.
  • the identification antigen to be sealed in the reaction container is preferably sealed in advance in a freeze-dried state. It is difficult to maintain the activity of the biomolecule constituting the identification antigen as described above. In particular, in the presence of water, it often denatures in a short time. In the present invention, it is possible to prevent deterioration before actual use by enclosing the identification antigen in a freeze-dried state in the reaction vessel in advance. Specifically, an aqueous solution of the identification antigen is added to the reaction vessel, frozen in a space of about ⁇ 20 ° C. to ⁇ 100 ° C., and freeze-dried and dried in a vacuum to obtain lyophilization. And can be stably enclosed.
  • identification antigen and the active ingredient can be mixed with a known amount of termite control agent which can be obtained only by lyophilizing only the identification antigen, and encapsulate in a freeze-dried state.
  • the reaction can be initiated simultaneously with the dissolution by adding the sample solution or solution on the spot to dissolve them.
  • a standard mixture consisting of the above-mentioned identification antigen and an active ingredient of a known amount of termite control agent is added to the reaction container in which the identification antigen is enclosed.
  • concentration of the active ingredient of the termite control agent in the sample liquid can be measured with high accuracy by using the termite control active ingredient as a comparison control without adding the active component of the termite control agent for measuring the ground.
  • a reaction vessel containing known amounts of the termite repellent active ingredient before and after that can be further prepared by preparing and comparing the reaction containers. It is possible to measure the concentration of the termiticide active ingredient in the sample liquid with high accuracy. In addition, the number of reaction containers in which a known amount of termite control active ingredient is enclosed is increased, and the concentration of the termite control active ingredient in the sample is unknown by comparison with a plurality of reference points. Even with this method, it is possible to measure the concentration of the active ingredient of the termiticide with high accuracy.
  • the identification antigen is lyophilised in advance and sealed in the reaction container, and in the reaction container, a diluted sample solution or a known amount of termite control agent is contained. It is also possible to dissolve with a standard solution having an active ingredient of Even if you use this method, you can start the reaction at the same time as dissolution by injecting and stirring the solution on site
  • a hapten compound derived from an active ingredient of a termite control agent is selected from bovine serum albumin (BSA), blood serum albumin (RSA), ovalbumin (OVA), keyhole limpet hemocyanin (KLH), thyroglobulin (TG) And after formation of a complex with a high molecular compound (protein) such as immunoglobulin, it is used as an antigen.
  • BSA bovine serum albumin
  • RSA blood serum albumin
  • OVA ovalbumin
  • KLH keyhole limpet hemocyanin
  • TG thyroglobulin
  • the method of forming the complex can be carried out by a known method, and is not particularly limited.
  • a complex is formed by reacting the carboxyl group of the hapten compound of fiprole or the hapten compound of imidacloprid with the functional group of the polymer compound, for example, by the mixed acid anhydride method or the active ester method.
  • a complex is formed by reacting the carboxyl group of the hapten compound of fiprole or the hapten compound of imidacloprid with the functional group of the polymer compound, for example, by the mixed acid anhydride method or the active ester method.
  • an antibody contained in blood such as Japanese cypress that has been immunized with the active ingredient of a termiticide is separated and purified!
  • Polyclonal antibody and antibody production There is a so-called monoclonal antibody which separates and purifies an antibody secreted by a functional cloned hybridoma.
  • any of the antibodies can be used.
  • Monoclonal antibodies The monoclonal antibodies are particularly preferred because they are highly selective for S imidacloprid.
  • the preparation method of the monoclonal antibody is not limited as long as it is a known method, but the monoclonal antibody secreted into ascites produced by inoculation of the hybridoma into the abdominal cavity of mouse can be purified using a protein A column or the like. .
  • the fixing member means a member having a rod-like portion formed of a resin capable of adsorbing proteins such as polyethylene 'polystyrene, which is easy to contact with a solution and has an antibody-immobilized portion having a large surface area. Preferred.
  • the fixing member having the antibody-immobilized portion be bonded to the sealing member, inserted into the reaction container, and have a structure capable of being fitted.
  • a configuration in which an antibody is immobilized on tip 3 can be mentioned.
  • a large surface area can be secured by forming the tip 3 into a vane shape.
  • the member thus immobilized can be combined with the immunoreaction container 4.
  • the lid 1 and the stick-shaped portion 2 are inserted into the reaction container in a state of being integrated. It is also possible to seal as it is.
  • lid 1 and the stick 2 of the sealing member may be configured to be separable!
  • the immune reaction container is sealed by the lid 1 and at the time of antigen-antibody reaction, the stick-shaped portion 2 is attached to the lid 1 and inserted into the reaction container and sealed by the lid 1 Stop. This operation makes it possible to stir the immune reaction container to promote the reaction.
  • the sealing member may have a configuration for sealing the reaction vessel 4 simply by the lid 1 as illustrated in FIG. 1 (B).
  • the freeze-dried identification antigen is sealed to ensure the stability of the identification antigen, and at the time of the reaction, a carrier 5 such as latex beads is enclosed in the reaction container 4 as an antibody fixing member.
  • a carrier 5 such as latex beads is enclosed in the reaction container 4 as an antibody fixing member.
  • the antibody is not directly immobilized on the sealing member, but is separated from the sealing member, and the bead carrier is used as a fixing member for the antibody.
  • a buffer solution containing the antibody may be placed on the sealing member and incubated.
  • the concentration of antibody in the buffer solution is usually about 0.01 ⁇ g ZmL to 10 gZmL.
  • the buffer known ones can be used without particular limitation.
  • the fixing member on which the antibody is immobilized is referred to as "antibody-immobilized carrier".
  • the antibody is immobilized in order to prevent nonspecific adsorption of contaminants in the sample on the surface of the carrier to affect the reaction, so that the surface part is not reactive with the antibody or the identification antigen. It is preferable to block by protein etc.
  • a blocking agent bovine serum albumin (BSA) or skimmed milk solution, or Block Ace (manufactured by Dainippon Sumitomo Pharma Co., Ltd.), etc. can be used.
  • Blocking is an excess of blocking agent solution This is carried out by bringing the antibody-immobilized carrier into contact, for example, incubating overnight at about 4 ° C., and then washing with a washing solution.
  • a buffer solution containing sodium chloride at physiological salt concentration can be used without limitation.
  • the antibody immobilized and blocked on the immobilized member can be stabilized by drying. Drying may be performed by any method such as vacuum lyophilization, vacuum drying, or air drying, which is better performed at a low temperature.
  • the sample prepared in the extraction unit is provided to the antigen-antibody reaction unit.
  • a sample is added to an immune reaction container in which a discrimination antigen is sealed, and an antigen-antibody reaction is initiated by inserting an immobilization material on which an antibody is immobilized.
  • the reaction container can be sealed by the sealing member combined with the inserted fixing member.
  • an immune reaction container containing an active ingredient of a known concentration of the insecticide and the identification antigen or an immunoreaction container containing only the identification antigen. Add a solution containing no active ingredient of the termite insecticide. By using this method, it is possible to more accurately determine the concentration of the termiticide active ingredient in the sample.
  • the antigen-antibody reaction should be carried out at a reaction temperature of 4 ° C. to 37 ° C. and a reaction time of 5 minutes to 2 hours.
  • the reaction vessel After completion of the reaction, wash the reaction vessel, the fixing member and the sealing member with tap water, purified water, buffer solution, etc. and then add to the reaction vessel a substrate solution that is colored by the enzyme of the identification antigen bound to the immobilized antibody.
  • a substrate solution that is colored by the enzyme of the identification antigen bound to the immobilized antibody.
  • an enzyme which is not particularly limited as long as it is a known substrate, for example, in the case of peroxidase, 3,3 ', 5,5'-tetramethylbenzidine may be used.
  • the concentration of the active ingredient of the pesticide is determined by detecting the change in the amount of color development that occurs after plating the substrate solution.
  • the determination can be easily performed by visually comparing the degree of color development by the active ingredient of a known concentration of termiticide.
  • the degree of color development can also be quantified by a spectrophotometer.
  • termite control agents can be measured at the site of application, they can be manipulated in the field. It is desirable to provide an easy-to-use measurement method.
  • a color is visually observed based on the surface color of the antibody-immobilized carrier. By detecting the change in quantity, it is possible to secure not only qualitative but also quantitative property of at least ppb order level.
  • the reaction vessel is a transparent reaction vessel 4 and the inner surface or half circumference of the reaction vessel 4 as illustrated in FIG. 1 (C).
  • a white member 6 to the outer surface of the reaction vessel 4 as illustrated in FIG. 1 (D), or as shown in FIG.
  • 2) as a white part and making the reaction vessel a transparent reaction vessel 4
  • color density may differ depending on temperature and time, and in particular, more accurate measurement can be made by comparing multiple reaction containers containing a known concentration of termite control active ingredient. Is possible. It can be measured on site and has excellent operability.
  • a spectrophotometer or the like as a detection means.
  • OPD o-ferendiamine
  • the absorbance at 490 nm is measured.
  • Other chromogenic substrates such as 3,3 ', 5,5'-tetramethyl benzidine can also be used. In this case, measure the absorbance at 650 nm.
  • absorbance at 450 nm may be measured.
  • alkaline phosphatase When alkaline phosphatase is used, for example, there is a method of measuring p-trophenyl phosphate as a chromogenic substrate.
  • concentration of the active ingredient in the termite control agent in the sample solution is determined using a calibration curve prepared from the relationship between the absorbance and the concentration of the reaction solution to which the termiticide active ingredient of known concentration was added. can do.
  • the active ingredient of the termite control agent can be measured by immunoassay in which the identification marker of the identification antigen is replaced with color latex or gold colloid.
  • a protein such as albumin is bound to the termite control active ingredient or its derivative, and a conjugate with this protein is immobilized on a color latex or gold colloid surface using a known method.
  • Prepared by The prepared identification antigen can be enclosed in an immune reaction container in the same manner as in the above-described example applied to the ELISA, and the effective component of termite control agent is produced by causing the antibody to react with the immobilized immobilization material. Can be measured.
  • color latex or gold colloid is bound to the immobilized antibody on the surface of the immobilizing member during an antigen-antibody reaction which requires a subsequent coloring operation. By visually judging the degree of coloration, the concentration of the active ingredient of the termiticide can be measured.
  • the measurement kit of the present invention may optionally include a dilution unit for diluting the sample solution prepared by the extraction unit to a predetermined ratio.
  • the dilution unit is preferable as a means for diluting to the optimum concentration range, as it is often difficult for ELISA to measure directly with high concentration of the above-mentioned solution.
  • Water is preferably used as the solution for dilution.
  • a final concentration of about 5 to 50% is preferable as a dilution rate of about 2 to 20 times.
  • the following device can be made as a specific example of a measurement kit for measuring the active ingredient of termite control agent in soil.
  • an extraction unit having an injection unit for adding a fixed amount of solvent is provided.
  • the soil to be measured can be placed, and the solvent can be added from the injection part.
  • the measurement kit further includes transfer means such as a tube pump for transferring the sample solution containing the active ingredient of the termite control agent from the extraction unit; the active ingredient of the antitermite control agent which reacts with the sample solution containing the active ingredient of the termite control agent
  • transfer means such as a tube pump for transferring the sample solution containing the active ingredient of the termite control agent from the extraction unit; the active ingredient of the antitermite control agent which reacts with the sample solution containing the active ingredient of the termite control agent
  • the reaction unit includes a fixed member on which the body is immobilized and a discrimination antigen; and a detection unit that detects a change in color amount of the sample solution reacted with the antigen antibody in the reaction unit.
  • the solvent may be automatically injected by putting a certain amount of soil. Alternatively, the solvent may be separately injected. Then mix in the extraction unit The active ingredient of the termiticide is extracted by stirring the material. Such supernatant of the extract is sent to the reaction unit by the transfer means, and a reaction solution is produced in the reaction unit by the antigen-antibody reaction. By measuring the absorbance of this reaction solution, it is possible to quantify the active ingredient of the termiticide in the soil.
  • a conjugate of Ushi serum albumin (BSA) and the fiprole hapten of (2) above was prepared as an immunogen using the active ester method.
  • the fipronil nopeptene solution prepared above was gradually added dropwise, and stirred at room temperature in the dark for 1.5 hours. After completion of the reaction, it was dialyzed against physiological phosphate buffer (PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0) at 4 ° C. for 2 days, and stored at 40 ° C. The conjugate of fiprole hapten thus obtained and BSA was used as an immunogen.
  • physiological phosphate buffer PBS, 10 mM phosphate buffer, 150 mM NaCl, pH 7.0
  • the immunogen prepared in (3) is dissolved in PBS so as to be 2 mg ZmL, and an equal amount of complete adjuvant (trade name: Freund's complete adjuvant; FCA) is mixed and equalized to form an emulsion, and 100 L of the mixture is added. 7-week-old female BalbZc mice were intraperitoneally administered. In a similar procedure to this, an adjuvant (trade name: Freund's incomplete adjuvant; FICA) was mixed with an equal volume of 0.5 mg ZmL of an immunogen 100 / z L for booster immunization every two weeks. After 4 rounds of immunization, blood was collected from the fundus and antibody titers in serum were confirmed by indirect competition.
  • FCA complete adjuvant
  • FICA Freund's incomplete adjuvant
  • Myeloma cells (P3 X 63 Ag 8. 653) cultured in advance were recovered, centrifuged, supernatant removed, and resuspended twice in serum-free DMEM medium.
  • Each cell number was counted, mixed so that the ratio of splenocytes to myeloma cells was 10: 1 to 7. 5: 1, centrifuged at 1500 rpm for 5 minutes, and the supernatant was aspirated and removed.
  • centrifuge tube LOOOrpm centrifuged for 5 minutes to completely aspirate the supernatant, HT medium (hypoxanthine as spleen cells become 2. 5 X 1 0 6 or ZML, thymidine, 10% fetal bovine The cells were suspended in serum-containing DM EM medium, 100 LZ-well aliquoted into a 96-well culture plate, and culture was started under humidified conditions at 37 ° C., 8% carbon dioxide gas.
  • HAT medium hypoxanthine, thymidine, aminopterin, 10% fetal calf serum-containing DMEM medium
  • HAT medium hypoxanthine, thymidine, aminopterin, 10% fetal calf serum-containing DMEM medium
  • the culture fluid was collected 10 days after the start of the culture, and the wells producing antibodies against fiproles were selected by indirect competitive inhibition ELISA method, and the culture scale was raised sequentially with 96 wells, 48 wells, and 24 wells.
  • the No. bridoma strain obtained as described above was cultured in DMEM containing 10% fetal bovine serum, and approximately 2 ⁇ 10 6 cells were injected intraperitoneally into BalbZc female Retiree mice, and ascites fluid was collected.
  • the ascites fluid obtained was subjected to IgG purification with a protein G column.
  • Fixation of the monoclonal antibody obtained in (5) to the fixing member was performed using an anti-mouse antibody of a secondary antibody.
  • the plate-like fixing member symbol 2 in FIG. 1 having a surface area of 5.2 cm 2 was placed in a solution of secondary antibody of 6 g Zml and left to stand still at 4 ° C. Next, it was washed with 10 mM phosphate buffer (PBS) containing 150 mM NaCl. The plate-like fixing member was placed in PBS containing 0.4% block air and allowed to stand at 20 ° C. for 1 hour. Furthermore, the stick was added to 5 g / ml of the monoclonal antibody solution obtained in (5), and the mixture was allowed to stand at 20 ° C. for 1 hour, washed and dried. In the following examples, fixed antibodies obtained in this way were used.
  • a conjugate of ushi serum albumin (BSA) and the imidaclopridonocephalus of (2) above was prepared as an immunogen using the active ester method.
  • 0.2 mmol of imidacloprid hapten prepared in (2) is dissolved in DMSO (1.0 mL), 0.3 mmol of N-hydroxysuccinimide and 0.3 mmol of 1-ethyl 3- (3 dimethylaminopropyl) carbodiimide are added, and the solution is added 3.5 Stir for hours. After the reaction, it was centrifuged at lOOOO rpm for 15 minutes to separate into a supernatant and a precipitate.
  • mice were used for immunization. 100 .mu.g of the imidaclopridono-peptide-BSA conjugate prepared in (3) was dissolved in 50 .mu.l of PBS, mixed with an equal volume of Freund's complete adjuvant, and inoculated subcutaneously into Balb Zc mice. Furthermore, after 4 weeks, the immunizing antigen prepared as described above was boosted using Freund's incomplete adjuvant. Also, at 6 weeks, the mouse tail vein was boosted with 30 g of the immunizing antigen dissolved in 180 L PBS.
  • mice with high anti-imidacloprid antibody activity in serum and myeloma cells were subjected to the method of Yamashita Shuji et al. Ed .: Interdisciplinary project. 1986) was fused by polyethylene glycol method and cultured.
  • the proliferation of cells was observed on the plate for assay that was coated with imidaclopridono- 1-BSA conjugate solution coated on a microplate and then diluted with Block Ace (“No. VK-25B” manufactured by Snow Brand Milk Products Co., Ltd., Code No. VK-25B). 50 each of the culture supernatants
  • the solution was added in an amount of ⁇ LZ and allowed to react at room temperature for 1 hour.
  • the No. bridoma strain obtained as described above was cultured in DMEM containing 10% fetal bovine serum, and approximately 2 ⁇ 10 6 cells were injected intraperitoneally into BalbZc female Retiree mice, and ascites fluid was collected.
  • the ascites fluid obtained was subjected to IgG purification with a protein G column.
  • Fixation of the monoclonal antibody obtained in (5) to the fixing member was performed using an anti-mouse antibody of a secondary antibody.
  • a plate-like fixing member symbol 2 in FIG. 1 having a surface area of 5.2 cm 2 was placed in a secondary antibody solution of 6 g / ml and allowed to stand still at 4 ° C. Next, it was washed with 10 mM phosphate buffer (PBS) containing 150 mM NaCl. The plate-like fixing member was placed in PBS containing 0.4% block and allowed to stand at 20 ° C. for 1 hour. Furthermore, the stick was placed in 5 g / ml of the monoclonal antibody solution obtained in (5), allowed to stand at 20 ° C. for 1 hour, washed and dried. In the following examples, fixed antibodies obtained in this manner were used.
  • Example 3 A fiplol measurement kit, to which ELISA is applied and measurement means consisting of each unit are combined, also specifically constitutes the reagent construction power as exemplified in Table 1 below.
  • the solution After dissolving fipronil standard reagent (manufactured by Wako Pure Chemical Industries, Ltd.) with methanol, the solution is diluted with purified water so as to be a 5% methanol solution, and the amount of 0.00050, 0.020, 0.20, 080, 0.20 ppm A standard dilution series was prepared.
  • the reaction vessel and the antibody stick were washed with tap water to remove the caries identifying antigen not bound to the immobilized antibody. After the residual solution of the washing solution was sufficiently removed, a coloring reagent was added to the reaction vessel, an antibody stick was attached, and a coloring reaction was performed for 10 minutes for each, and then the absorbance at 650 nm was determined. As a result, as shown in FIG. 2, it was found that fipronil can be measured with a substantially linear standard curve up to 0. 0050-0. 080 ppm. Therefore, it was considered that visual judgment would be possible within this measurement range.
  • the fipronil measurement kit combining the measurement means consisting of each unit is shown in the following table.
  • the reagent composition is as exemplified in 2.
  • Sample soil (containing 1 ppm of fibrils and 10% of organic matter) lg was weighed, transferred to an extraction vessel, stirred in 5 mL of 50% methanol for 1 minute, and the fibril contained therein was extracted. . Furthermore, tap water was added to the extraction vessel to make a 50 mL mixture, and the supernatant was used as the sample solution.
  • the reaction vessel and the antibody stick were washed with tap water to remove the caries identifying antigen not bound to the immobilized antibody. After the residual solution of the washing solution was sufficiently removed, a coloring reagent was added to the reaction vessel, an antibody stick was attached, and a coloring reaction was performed for 10 minutes for each, and then the absorbance at 650 nm was determined.
  • a calibration curve was prepared using a standard solution, and the concentration of fipolles recovered from the sample soil was determined. As a result, it was possible to measure fipronil in the soil with a good fiprole concentration of 1 ppm.
  • Example 5 [0153] Next, specific examples of a measurement kit and a measurement method for visually determining fipronil in soil are listed.
  • the Fiprole measurement kit in which the measurement means consisting of each unit are combined also has the reagent constitution power as exemplified in Table 3 below.
  • Sample soil (containing 1 ppm of fibrils and 10% of organic matter) lg was weighed, transferred to an extraction vessel, stirred in 5 mL of 50% methanol for 1 minute, and the fibril contained therein was extracted. . Furthermore, tap water was added to the extraction vessel to make a 50 mL mixture, and the supernatant was used as the sample solution.
  • the reaction vessel and the antibody stick were washed with tap water to remove the caries identifying antigen not bound to the immobilized antibody. After the residual solution of the washing solution was sufficiently removed, a coloring reagent was added to the reaction vessel, and an antibody stick was attached, and after making each color react for 10 minutes, visual judgment was made. As a result, the data Although it does not show, the sample soil shows the same coloration level as the positive control (including fiprole 0. 02 ppm; equivalent to 1. O ppm as fipronil concentration in the soil), and it is in the soil based on 1 ppm fiprole concentration. We know that we can determine the fipronil concentration of
  • the Fiprole measurement kit in which the measurement means consisting of each unit are combined also has the reagent composition as exemplified in Table 4 below. The procedure for visually determining the concentration of fipronil in soil using this measurement kit will be described.
  • the fipronil used here is formulated as microbe.
  • Sample soil (microcapsulated fipolol (1 ppm equivalent to fipolol) and 10% organic substance) Weigh and transfer to an extraction vessel and stir in 3 mL of 100% ethanol for 1 minute And extracted fipronil contained therein. Furthermore, tap water was added to the extraction vessel to make a 50 mL mixture, and the supernatant was used as a sample solution.
  • the lysis reagent ImL was added to the reaction vessels A and B, and the sample solution ImL was added to the reaction vessel C and sealed in advance to dissolve and mix the identification antigen (in the case of B, containing fiproles). Immediately, the antibody stick was attached to the reaction vessel, and an antigen-antibody reaction was carried out at 25 ° C. for 10 minutes. (3) Detection of change in color amount
  • the reaction vessel and the antibody stick were washed with tap water to remove the caries identifying antigen not bound to the immobilized antibody. After the residual solution of the washing solution was sufficiently removed, a coloring reagent was added to the reaction vessel, and an antibody stick was attached, and after making each color react for 10 minutes, visual judgment was made. As a result, the data is not shown, but the sample soil shows the same coloration level as the positive control (including fibral 0. 02 ppm; equivalent to 1. O ppm as fipronil concentration in the soil), and the fiprol concentration l ppm It has been found that fipronil concentration in soil can be qualitatively determined as a standard o
  • Example 4 the detection of the change in color amount may be carried out by visual determination.
  • the color reaction may be performed by determining the absorbance at 650 nm.
  • the imidacloprid measurement kit in which the ELISA is applied as described above and the measurement means consisting of each unit are combined also specifically constitutes the reagent constitution power as exemplified in Table 5 below.
  • the solution After dissolving imidacloprid standard reagent (manufactured by Wako Pure Chemical Industries, Ltd.) with methanol, the solution is diluted with purified water so as to be a 5% methanol solution, and 0.20, 0.20, 0. 0008, 0. 0016, 0. 0031, 0 .
  • Antigen-antibody reaction 1 mL of each standard solution was added to the reaction vessel to dissolve and mix the pre-enclosed, discriminating antigen. Immediately, the antibody stick was attached to the reaction vessel, and antigen-antibody reaction was performed at 25 ° C for 10 minutes o
  • the reaction vessel and the antibody stick were washed with tap water to remove the identification antigen which did not bind to the immobilized antibody. After the residual solution of the washing solution was sufficiently removed, a coloring reagent was added to the reaction vessel, an antibody stick was attached, and a coloring reaction was performed for 10 minutes for each, and then the absorbance at 650 nm was determined. As a result, it was found that imidacloprid can be measured with a linear standard curve ranging from 0. 002 ppm to 0. 05 ppm as shown in FIG. Therefore, it was considered that visual judgment could be possible within this measurement range.
  • the imidacloprid measurement kit combining the measurement means consisting of each unit also constitutes a reagent composition as exemplified in Table 6 below.
  • the measurement kit was actually used to measure imidacloprid in the soil.
  • the reaction vessel and the antibody stick were washed with tap water to remove the caries identifying antigen not bound to the immobilized antibody. After the residual solution of the washing solution is sufficiently removed, the color developing reagent is added to the reaction vessel, the antibody stick is attached, and the color reaction is allowed for 10 minutes. Then, the antibody stick is removed from the reaction vessel, and the color reaction in the reaction vessel is carried out. For the liquid, the absorbance at 650 nm was determined using a spectrophotometer.
  • a standard curve was prepared using a standard solution, and the concentration of imidacloprid recovered from the sample soil was determined. As a result, the imidacloprid concentration was as good as 1 ppm, and imidacloprid in the soil could be measured.
  • the imidacloprid measurement kit in which the measurement means consisting of each unit are combined also has the reagent constitution power as exemplified in Table 7 below.
  • the procedure for visually determining the idacloprid concentration in soil using this measurement kit will be described.
  • sample solution Sample soil (containing 1 ppm imidacloprid and 10% organic matter) 1 g was weighed, transferred to an extraction vessel, stirred in 5 mL of 50% methanol for 1 minute, and the imidacloprid contained therein was extracted. Further, tap water was added to the extraction vessel to make a 50 mL mixed solution, and the supernatant was used as a sample solution.
  • the reaction vessel and the antibody stick were washed with tap water to remove the caries identifying antigen not bound to the immobilized antibody. After the residual solution of the washing solution was sufficiently removed, a coloring reagent was added to the reaction vessel, and an antibody stick was attached, and after making each color react for 10 minutes, visual judgment was made. As a result, the data is not shown, but the sample soil shows the same coloration level as the positive control (containing 0.20 ppm of imidacloprid; equivalent to 1. Oppm concentration of imidacloprid in the soil), and the concentration of imidacloprid was 1 ppm It was found that the imidacloprid concentration of can be determined qualitatively.
  • the imidacloprid measurement kit combining the measurement means consisting of each unit also constitutes the reagent constitution power as exemplified in Table 8 below. The procedure for visually determining the imidacloprid concentration in soil using this measurement kit will be described.
  • the imidacloprid used herein is formulated as a microcapsule.
  • Antibody Stick Anti-imidacloprid pile body is cut to H
  • Sample soil Weigh 1g of soil (microcapsule formulated imidacloprid (equivalent to lppm in imidacloprid) and 10% organic matter), transfer to an extraction vessel and stir in 3mL of 100% ethanol for 1 minute, The imidacloprid contained in it was extracted. Furthermore, tap water is put into the extraction vessel to make a 50 mL mixture, and the supernatant is used as a sample solution.
  • the reaction vessel and the antibody stick were washed with tap water to remove the caries identifying antigen not bound to the immobilized antibody. After the residual solution of the washing solution was sufficiently removed, a coloring reagent was added to the reaction vessel, and an antibody stick was attached, and after making each color react for 10 minutes, visual judgment was made. As a result, the data is not shown, but the sample soil shows the same coloration level as the positive control (containing 0.20 ppm of imidacloprid; equivalent to 1. Oppm concentration of imidacloprid in the soil), and the concentration of imidacloprid was 1 ppm It was found that the imidacloprid concentration of can be determined qualitatively.
  • Example 8 the detection of the change in color quantity is carried out by visual judgment.
  • the color change may be determined by determining the absorbance at 650 nm.

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Abstract

L’invention concerne un procédé de dosage de l’ingrédient actif d’un agent anti-termites permettant de doser l’ingrédient actif de l’agent anti-termites au moyen d’un procédé d’immunodosage tout en assurant des conditions de manipulation favorables au niveau du site d’application. L’invention concerne également un kit associé à ce procédé de dosage. Le kit de dosage de l’ingrédient actif de l’agent anti-termites au moyen d’un procédé d’immunodosage comprend : (1) un module d’extraction servant à extraire d’un sujet échantillonné à doser l’ingrédient actif de l’agent anti-termites à l’aide d’un solvant ; et (2) un module de réaction comprenant un récipient de réaction (4) contenant un antigène discriminant, un élément immobilisateur (2) servant à immobiliser un anticorps représentant l’ingrédient actif de l’agent anti-termites, et un élément d’étanchéité (1) qui peut être inséré dans le récipient de réaction. Le kit de dosage est caractérisé en ce qu’une réaction antigène-anticorps y est mise œuvre par contact avec la solution échantillon ou une solution échantillon diluée. Le kit de dosage peut éventuellement comprendre : (3) un module de détection servant à détecter, à l’œil nu ou par voie optique, une variation de la concentration de l’ingrédient actif de l’agent anti-termites dans le sujet échantillonné due à la réaction susmentionnée ; et (4) un module de dilution servant à diluer la solution échantillon à un rapport de dilation déterminé.
PCT/JP2006/324930 2005-12-16 2006-12-14 Kit de dosage de l’ingredient actif d’un agent anti-termites au moyen d’un procede d’immunodosage Ceased WO2007069681A1 (fr)

Priority Applications (3)

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AU2006325990A AU2006325990B2 (en) 2005-12-16 2006-12-14 Kit for assaying active ingredient of termite controlling agent by using immunoassay method
EP06834684A EP1965209A4 (fr) 2005-12-16 2006-12-14 Kit de dosage de l ingredient actif d un agent anti-termites au moyen d un procede d immunodosage
US12/097,627 US8323904B2 (en) 2005-12-16 2006-12-14 Kit for measurement of termite insecticide active ingredient by immunoassay method

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JP2005-363166 2005-12-16
JP2005363166 2005-12-16
JP2006309332A JP2007187650A (ja) 2005-12-16 2006-11-15 免疫測定法を用いたフィプロニルの測定キット
JP2006-309332 2006-11-15
JP2006332073A JP2008145240A (ja) 2006-12-08 2006-12-08 免疫測定法を用いたイミダクロプリドの測定キット
JP2006-332073 2006-12-08

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CN105301233A (zh) * 2015-08-21 2016-02-03 贵州勤邦食品安全科学技术有限公司 检测茶叶中吡虫啉残留的酶联免疫试剂盒及使用方法
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